CN105176816B - A kind of microvascular liver chip and its preparation and application based on cell aggregation - Google Patents
A kind of microvascular liver chip and its preparation and application based on cell aggregation Download PDFInfo
- Publication number
- CN105176816B CN105176816B CN201510720578.9A CN201510720578A CN105176816B CN 105176816 B CN105176816 B CN 105176816B CN 201510720578 A CN201510720578 A CN 201510720578A CN 105176816 B CN105176816 B CN 105176816B
- Authority
- CN
- China
- Prior art keywords
- cell
- liver
- culture
- microvascular
- chip
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
- C12M23/16—Microfluidic devices; Capillary tubes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
- C12M25/06—Plates; Walls; Drawers; Multilayer plates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
- C12M25/14—Scaffolds; Matrices
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5014—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
- G01N33/5067—Liver cells
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Immunology (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Molecular Biology (AREA)
- Genetics & Genomics (AREA)
- General Engineering & Computer Science (AREA)
- Sustainable Development (AREA)
- Cell Biology (AREA)
- Toxicology (AREA)
- Medicinal Chemistry (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Food Science & Technology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Gastroenterology & Hepatology (AREA)
- Dispersion Chemistry (AREA)
- Clinical Laboratory Science (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a kind of microvascular liver chip based on cell aggregation, including the microvasculature positioned at upper strata, middle class blood vessel endothelium barrier system and the liver organ many cells co-culture system of lower floor, microvasculature and liver organ many cells co-culture system are respectively arranged in respective chip base；Wherein, the microvasculature includes the curved vessel being made up of some choked flow fence interlaced arrangements, and the curved vessel two ends are respectively equipped with microvascular import and microvascular outlet；The class blood vessel endothelium barrier system is made up of perforated membrane；The liver organ many cells co-culture system includes cell aggregation enrichment region and various kinds of cell and co-cultures area, and is respectively arranged with two ends culture systems import and culture systems outlet.The present invention can carry out the preparation of liver diseases model and the research of pharmacokinetics and pharmaceutical activity, medicine low consumption small with amount of samples, the characteristics of portable, economic, efficient, accurate.
Description
Technical field
The present invention relates to a kind of microvascular liver chip based on cell aggregation and its preparation and application, belong to micro-
Fluid chip fabrication design and biomedicine technical field.
Background technology
Safely and effectively medicine is a long-term, difficult and expensive process for research and development.Cell culture and zoopery are existing
For the two kinds of experiment porch for widely using and extremely relying in medical science and study of pharmacy.Medicine enter it is preclinical will be by vitro
Experiment and animal transition experiment detect effect of the medicine to cultured cell in vitro and animal body, and be repeated pharmacodynamics,
Pharmacokinetics, the qualitative and quantitative forecast of toxicology.But a major reason of medicament research and development failure is to be currently used in
The difference that the model of assessing drug actions and the disease model presence of people can not be avoided.Under normal circumstances, cell culture pattern is difficult mould
Anthropomorphic body microenvironment, meanwhile, normal and disease physiology course is analyzed using animal model, it is not only costly, time-consuming,
And in the presence of arguement ethically.The experimental drug verified by this two classes experiment porch is washed in a pan after clinical test is entered
Eliminate, or drug effect is low or toxic side effect is strong, causes the huge waste of man power and material in medicament research and development.
Organ chip can break through the limitation of cell culture and model animal experiment.It is one and is based on multi-channel fluid core
The Three-dimensional cell culture system of piece, is formed by the cell culture partitioned set of multiple simulated human tissues and organ environment.Each
Subregion is attached by the bionical circulatory system.The size of microchannel can be suitable with cell size, can accurately control micro-
The situation of the factors such as composition, the temperature of environment, as far as possible analog cell epimatrix, the reliability and operability of Enhancement test.
Can design simultaneously different two dimension or three-dimensional structure and Precision Machining into the devices such as microelectrode realize the culture of cell, positioning,
In order, a variety of functions such as patterning and detection.The environment that organism is simulated on chip carries out the training of cell, tissue and organ
Support, the biological behaviour studied and control cell in vitro in incubation, and in terms of drug development, medical diagnosis on disease and treatment
It is with a wide range of applications.The research of human organ chip is still in the starting stage in the world, but American-European-Japanese etc. national
Different types of brain chip, lung chip, heart chip, kidney chip, liver chip, spleen chip, intestines chip etc. are developed a variety of
The chip of organ, the research such as drug screening is carried out to which animal model is simulated and substituted for organ dysfunction.
Liver is the most important removing toxic substances organ of human body, is also the major organs that medicine realizes conversion and metabolism, builds simulation
Drug is screened and provides good platform by the liver chip of liver organ function and structure.However, current liver chip class
Type is very few, only other to simulate class blood vessel endothelium barrier and part microenvironment, or liver micro-loop difficult to realize
The system simulation in border is simultaneously widely used in the research of drug screening.Inherently one splendid liver cell reactor of liver, liver
Dirty basic structure and functional unit are lobuli hepatis, and its interior liver cell does not only reach the requirement of quantity and density, polarity is also presented
Ordered arrangement；Liver cell is in a kind of three-dimensional environment, liver cell and the hepatic sinusoidal endothelial cells around it, and liver is starlike thin
Born of the same parents, Kupffer cell, and extracellular matrix etc. interact, and cell-cell interaction also contributes to adjust growth and the work(of cell
Can differentiation.Blood vessel and biliary system provide oxygen and nutriment for liver cell in liver, and medicine also realizes generation in liver
Thank and convert, while taking away the metabolic waste of liver cell.
The content of the invention
For above-mentioned the deficiencies in the prior art, it is an object of the invention to provide a kind of microvascular liver based on cell aggregation
Dirty chip and its preparation and application, the organ chip simulate the complicated a variety of microenvironment key elements of liver, solve tradition
Drug screening in using cell and animal model inherent shortcoming, solve the structure that existing liver cell cultivating system is present
It is single, and the few shortcoming of analog functuion, it can effectively carry out the implantation of positioning successively of many cells and cultivate, and carry out the peace of medicine
Full property and efficiency evaluation.
To achieve the above object, the present invention uses following technical scheme：
A kind of microvascular liver chip based on cell aggregation, including the microvasculature positioned at upper strata, middle class
Blood vessel endothelium barrier system and the liver organ many cells co-culture system of lower floor, microvasculature and liver organ many cells are total to
Culture systems are respectively arranged in respective chip base；Wherein, the microvasculature is included by some choked flow fence interlaced arrangements
The curved vessel of composition, the curved vessel two ends are respectively equipped with microvascular import and microvascular outlet；The class blood vessel endothelium
Barrier system is made up of perforated membrane；The liver organ many cells co-culture system includes cell aggregation enrichment region and a variety of thin
Born of the same parents co-culture area, and are respectively arranged with two ends culture systems import and culture systems outlet, and liver organ many cells are co-cultured
The interior zone that system is located at culture systems import is provided with two shunting fence, wherein, cell aggregation enrichment region is by multiple
Some gaps, multiple crescent moon groove-like structure longitudinal arrangements are provided with crescent moon channel-shaped structure composition, each crescent shape structure
Into several columns, and the several columns interlaced arrangement, the opening of all crescent moon groove-like structures is towards culture systems import.
The chip base is dimethyl silicone polymer（PDMS）, polymethyl methacrylate（PMMA）, glass, makrolon
（PC）, polytetrafluoroethylene (PTFE), nitrocellulose, biomembrane, the one or more of silicon chip.
Cell adherence and culture matrix used in the liver organ many cells co-culture system are collagen, fiber egg
White glue, extracellular matrix protein, BSA albumen, fibroin albumen, gelatin, chitosan, arginine-glycine-aspartic acid（RGD）、
Matrigel（Matrigel）, sodium alginate, different Methacrylamide（NIPAM）, polyethylene glycol（PEG）Or polyethylene glycol acrylic acid
Ester（PEGDA）One or more, prioritizing selection is collagen.
The scarcely perceptible pulse tube inlet and microvascular exit passageway depth, choked flow fence height and curved vessel of the microvasculature
Depth is 100 μm；The width of choked flow fence is 20 μm, is shaped as semicircle chamfering, and co-culture with liver organ many cells
The position of crescent moon groove-like structure is corresponding in system；Curved vessel is round and smooth passage, and channel width is 600 μm, and number of bends is
20。
The material of the perforated membrane is dimethyl silicone polymer（PDMS）, Transwell PET or PC films one
Kind, thickness is 10 μm, and aperture is 0.4,1,3,5,8 μm, and prioritizing selection aperture is 1 μm of PDMS flexible membranes.
The culture systems entrance and culture systems exit passageway depth of the liver organ many cells co-culture system, shunting
The height of fence and crescent moon groove-like structure is 100 μm；Shunting fence is round and smooth arc, is alignd with intake channel, and equidistant right
Claim arrangement；The number of crescent moon groove-like structure is 80, and its outer toroid radius is 350 μm, and interior annular radius is 150 μm, crescent
Gap in shape structure is four, and uniformly diverging arrangement, and its width is 20 μm, 100 μm of depth, interior annular opening air line distance
For 100 μm, it is 100 μm that crescent moon groove-like structure, which spaces between distance,.
A kind of preparation method of the microvascular liver chip based on cell aggregation, comprises the following steps：
（1）The mask plate of organ chip is designed and drawn with computer aided design software, each layer chip of accurate Drawing
Micro-structural and microchannel figure；
（2）By the method for micro-processing technology, particularly photoengraving, prepare bilevel mask, prepare containing
Micro-structural and the chip of microchannel；
（3）By the microvasculature chip on upper strata, middle perforated membrane and the liver organ many cells of lower floor co-culture system
System is alignd, and is pressurizeed, and carries out plasma bonding, forms the micro-structural and microchannel network of 3 D stereo；
（4）Different cell categories are planted by microvascular import and culture systems intake channel successively, and carry out it is adherent and
Three-dimensional aggregates culture, and carry out liver parenchymal cell pattern and feature（Albumin is secreted, urea synthesizing, P450 enzymatic activitys
Deng）Evaluate；
（5）Medicine is passed through by the upper and lower passage successively and carries out medicine hepatotoxicity and pharmaceutical activity evaluation（Medicine
Long-term and short-term hepatotoxicity, and medicine IC50 values）.
Vascular endothelial cell, cell are planted in the closed area of microvasculature and the formation of class blood vessel endothelium barrier system
Density is 1x106Individual/mL, adhere-wall culture 2 ~ 4 hours forms vascular endothelial cell layer.
Various kinds of cell is planted successively in liver organ many cells co-culture system, and liver cell aggregation passes through culture first
System inlets passage enters the crescent moon groove-like structure of cell aggregation enrichment region, and enrichment and growth grows for three-dimensional aggregates；It is many
Plant cell and co-culture area by culture systems intake channel plantation co-cultured cell species, co-cultured cell species includes liver star
Cell, Kupffer cell, horn cell, and fibroblast, cell density are 1x106Individual/mL, adhere-wall culture 2 ~ 4 hours.
A kind of application method of the microvascular liver chip based on cell aggregation,：When carrying out drug evaluation, medicine leads to
Cross microvasculature and enter chip, then penetrate class blood vessel endothelium barrier system, the liver organ many cells for entering lower floor are total to
Culture systems, so as to evaluate the toxic action of its liver and carry out the metabolism and conversion of medicine；Or, medicine is also by culture
System inlets are directly entered liver organ many cells co-culture system, directly enter with hepatic parenchymal cells aggregation and co-cultured cell
Row contacts and realizes metabolism and convert, and verifies the hepatotoxicity and medicine vigor of medicine.
The beneficial effects of the invention are as follows：
（1）Liver chip constructs microvasculature, and it is intravascular based on perforated membrane and endothelial cell to be prepared for class
Skin barrier system, effectively simulates the microvascular and endothelial barrier function inside hepatic sinusoid, can supply nutrition and oxygen.
（2）Liver chip carries out three-dimensional aggregates by many gap crescents design enrichment hepatic parenchymal cells aggregation
Long-term cultivation, effectively realize high density, positioning and the dimensional culture of cell inside liver, contribute to the information for realizing cell to hand over
Stream and mutual promoting action.
（3）Liver chip is designed by many gap crescents and plants other liver kinds around liver cell aggregation rich region
The cell of class, realizes the co-cultivation of various kinds of cell, effectively simulates cell type and cell various inside actual liver organ
Between interaction.
（4）Liver chip can realize the long-term cultivation of hepatic parenchymal cells, and carry out effective drug evaluation application, transparent
Material can monitor the biological behaviour of cell, tissue and organ on-line, so as to be cell-drug interaction and drug screening
There is provided a brand-new technology platform.
Brief description of the drawings
Fig. 1 is the structural representation of the present invention；
Fig. 2 is the structural representation of microvasculature；
Fig. 3 is the structural representation of class blood vessel endothelium barrier system；
Fig. 4 is the structural representation of liver organ many cells co-culture system；
Fig. 5 is the enlarged drawing at A positions in Fig. 4；
Fig. 6 is the cellular localization distribution situation of the microvascular liver chip based on cell aggregation,（a）Exist for adherent growth
On perforated membrane vascular endothelial cell layer, and carried out green fluorescence Calcein-AM dyeing,（b）It is three-dimensional for liver parenchymal cell
Aggregation enrichment and growth, and carried out blue-fluorescence Hochest33342 nuclear targetings,（c）It is common in many cells for adherent growth
The MEC in region is cultivated, and has carried out green fluorescence Calcein-AM dyeing.
In figure, 1- microvasculatures, 2- class blood vessel endothelium barrier systems, 3- liver organ many cells co-culture systems, 4-
Choked flow fence, 5- curved vessels, 6- microvascular imports, the outlet of 7- microvasculars, 8- perforated membranes, the enrichment of 9- liver cells aggregation
Area, 10- various kinds of cell co-cultures area, and 11- culture systems are exported, 12- culture systems imports, 13- shunting fence, 14- gaps,
15- crescent moon groove-like structures.
Embodiment
The present invention is further described below in conjunction with the accompanying drawings.
It is a kind of microvascular liver chip based on cell aggregation as Figure 1-5, it is characterised in that：Including positioned at upper
The microvasculature 1 of layer, middle class blood vessel endothelium barrier system 2 and the liver organ many cells co-culture system 3 of lower floor, it is micro-
Vascular system 1 and liver organ many cells co-culture system 3 are respectively arranged in respective chip base；
Wherein, such as Fig. 2, microvasculature 1 includes the curved vessel 5 being made up of some interlaced arrangements of choked flow fence 4, described
The two ends of curved vessel 5 are respectively equipped with microvascular import 6 and microvascular outlet 7；
Such as Fig. 3, class blood vessel endothelium barrier system 2 is made up of perforated membrane 8；
Such as Fig. 4, liver organ many cells co-culture system 3 includes cell aggregation enrichment region 9 and various kinds of cell is co-cultured
Area 10, and culture systems import 12 and culture systems outlet 11 are respectively arranged with two ends, liver organ many cells co-culture system
The interior zone that system 3 is located at culture systems import 12 is provided with two shunting fence 13, and the purpose is to allow the cell being passed through logical
It is dispersed in road to open, prevent edge from there is no cell, directly pass through chip from center-aisle, wherein, cell aggregation enrichment region 9
It is made up of multiple crescent moon groove-like structures 15, such as Fig. 5, some gaps 14, Duo Geyue is provided with each crescent shape structure 15
The longitudinal arrangement of tooth groove-like structure 15 is into several columns, and the several columns interlaced arrangement, the equal court of opening of all crescent moon groove-like structures 15
Culture systems import 12.
Chip base is dimethyl silicone polymer（PDMS）, polymethyl methacrylate（PMMA）, glass, makrolon（PC）、
Polytetrafluoroethylene (PTFE), nitrocellulose, biomembrane, the one or more of silicon chip.
Cell adherence and culture matrix used in liver organ many cells co-culture system 3 are collagen, fibrin
Glue, extracellular matrix protein, BSA albumen, fibroin albumen, gelatin, chitosan, arginine-glycine-aspartic acid（RGD）, base
Matter glue（Matrigel）, sodium alginate, different Methacrylamide（NIPAM）, polyethylene glycol（PEG）Or polyethylene glycol acrylate
（PEGDA）One or more, prioritizing selection is collagen.
The scarcely perceptible pulse tube inlet 6 and microvascular of microvasculature 1 export 7 channel depths, the height of choked flow fence 4 and curved vessel
5 depth are 100 μm；The width of choked flow fence 4 is 20 μm, is shaped as semicircle chamfering, and train altogether with liver organ many cells
The position for supporting crescent moon groove-like structure 15 in system 3 is corresponding；Curved vessel 5 is round and smooth passage, and channel width is 600 μm, curved
Number of tracks is 20.
The material of perforated membrane 8 is dimethyl silicone polymer（PDMS）, Transwell PET or PC films one kind, it is thick
Spend for 10 μm, aperture is 0.4,1,3,5,8 μm, prioritizing selection aperture is 1 μm of PDMS flexible membranes.
The culture systems entrance 11 and culture systems of liver organ many cells co-culture system 3 export 12 channel depths, point
The height for flowing fence 13 and crescent moon groove-like structure 15 is 100 μm；Shunting fence 13 is round and smooth arc, is alignd with intake channel,
And be equidistantly arranged symmetrically；The number of crescent moon groove-like structure 15 is 80, and its outer toroid radius is 350 μm, and interior annular radius is 150
μm, the gap 14 in crescent moon groove-like structure 15 is four, and uniformly diverging arrangement, and its width is 20 μm, 100 μm of depth, inner circle
Ring opening air line distance is 100 μm, and it is 100 μm that crescent moon groove-like structure 15, which spaces between distance,.
The preparation method of the above-mentioned microvascular liver chip based on cell aggregation, comprises the following steps：
（1）Use computer aided design software（Such as Auto-CAD or Solidwork）Design and draw covering for organ chip
Diaphragm plate, the micro-structural and microchannel figure of each layer chip of accurate Drawing；
（2）By the method for micro-processing technology, particularly photoengraving, prepare bilevel mask, prepare containing
Micro-structural and the chip of microchannel；
（3）By the microvasculature chip on upper strata, middle perforated membrane and the liver organ many cells of lower floor co-culture system
System is alignd, and is pressurizeed, and carries out plasma bonding, forms the micro-structural and microchannel network of 3 D stereo；
（4）Different cell categories are planted by microvascular import and culture systems intake channel successively, and carry out it is adherent and
Three-dimensional aggregates culture, and carry out liver parenchymal cell pattern and feature（Albumin is secreted, urea synthesizing, P450 enzymatic activitys
Deng）Evaluate；
（5）Medicine is passed through by the upper and lower passage successively and carries out medicine hepatotoxicity and pharmaceutical activity evaluation（Medicine
Long-term and short-term hepatotoxicity, and medicine IC50 values）.
Vascular endothelial cell is planted in the closed area of microvasculature 1 and the formation of class blood vessel endothelium barrier system 2, carefully
Born of the same parents' density is 1x106Individual/mL, adhere-wall culture 2 ~ 4 hours forms vascular endothelial cell layer.
Various kinds of cell is planted successively in liver organ many cells co-culture system 3, and liver cell aggregation passes through training first
The crescent moon groove-like structure 15 that the passage of system inlets 12 enters cell aggregation enrichment region 9 is supported, and enrichment and growth is three-dimensional aggregates
Growth；Various kinds of cell co-cultures area 10 and plants co-cultured cell species, co-cultured cell kind by the passage of culture systems import 12
Class includes stellate cells, Kupffer cell, horn cell, and fibroblast, and cell density is 1x106Individual/mL, it is adherent
Culture 2 ~ 4 hours.
The application method of the above-mentioned microvascular liver chip based on cell aggregation is：When carrying out drug evaluation, medicine
Chip is entered by microvasculature 1, class blood vessel endothelium barrier system 2 is then penetrated, how thin the liver organ for entering lower floor is
Born of the same parents' co-culture system 3, so as to evaluate the toxic action of its liver and carry out the metabolism and conversion of medicine；Or, medicine also leads to
Cross culture systems import 12 and be directly entered liver organ many cells co-culture system 3, with hepatic parenchymal cells aggregation and co-cultivation
Cell is directly contacted and realizes metabolism and convert, and verifies the hepatotoxicity and medicine vigor of medicine.
Such scheme is described further below in conjunction with specific embodiment.It should be understood that these embodiments are to be used to illustrate
The present invention and be not limited to limit the scope of the present invention.The implementation condition used in embodiment can be done further according to actual conditions
Adjustment, unreceipted implementation condition is usually the condition in normal experiment.
The design and preparation of microvascular liver chip of the embodiment 1 based on cell aggregation
Use computer aided design software（Such as Auto-CAD or Solidwork）Design and drafting are based on cell aggregation
Microvascular liver chip micro-structural and microchannel figure.The spin coating photoresist on egative film is used first, it is certainly aobvious by photoetching
Movie queen forms photoetching sealing rubber die, touches tool by the silicon color sensor fabrication techniques silicon in micro electro mechanical processing, numerical control CNC systems can also be used
System processing prepares micro-structural and the microchannel of chip.By dimethyl silicone polymer（PDMS）Performed polymer mixed with curing agent, pass through
Above and below being cast in after the steps such as ultrasound, stirring, vacuum outgas on the mould of layers of chips, in 70 ~ 80 DEG C of thermal responses 1 ~ 3 hour or
65 DEG C are stayed overnight thermal response, natural cooling.By dimethyl silicone polymer（PDMS）Chip is carefully taken off from mould, and two by above and below
Layer chip is alignd with being placed in the clean perforated membrane of centre, bonded, plasma is bonded, and is formed with microvasculature, class
The liver chip of blood vessel endothelium barrier system and liver many cells co-culture system.
The cell that obtains of the liver chip many cells co-culture system of embodiment 2 is implanted into and localized cell culture
Import and the entrance of liver many cells co-culture system passage are closed, the inlet and outlet of microvasculature is opened,
Via the import of microvasculature, culture fluid of endothelial cell is slowly flowed through to microchannel and entered in the curved vessel of microvasculature
Portion, exquisiteness culture 2 ~ 4 hours treats that endothelial cell adherent growth on middle perforated membrane, forms class blood vessel endothelium barrier.Close
The inlet and outlet of microvasculature, opens the export and import of liver organ many cells co-culture system, is prepared by sessile drop method
Liver cell aggregation nutrient solution be slowly injected into liver many cells co-culture system cavity, because gap width is smaller, training
Nutrient solution and individual cells are easy to pass through, and the diameter of cell aggregation is than larger, and cell aggregation, which is known from experience, is constantly enriched in the moon
Alveolus inside configuration, forms bigger cell aggregation, and according to the progress of the shape of crescent moon slot structure three-dimensional culture and growth,
The cell or cell aggregation at large for obtaining and being enriched in inside crescent is washed with fresh medium.Continue a variety of livers
The nutrient solution of the co-cultured cells such as astrocyte spongiocyte, Kupffer cell, and fibroblast slowly flows through microchannel
Into in the cavity of liver many cells culture systems, quiescent culture 2 ~ 4 hours, adherent growth.After cell attachment is firm, carry out
Continuous nutrient solution supply.
The pharmaceutical activity of embodiment 3 and safety detection
After cytotostatic grows and breeds, hepatotoxicity and the pharmaceutical activity evaluation of medicine are carried out.Medicine is by different dense
Degree is dissolved in cell culture fluid, is flowed slowly into via microvasculature feeder connection inside the curved vessel of microvasculature,
Medicine passes through the perforated membrane cellular layer of class blood vessel endothelium barrier system, and the liver organ many cells for entering lower floor co-culture system
System, and via the metabolism and conversion of liver parenchymal cell, liver cell morphology characterization and cellular functional activity are carried out with this（As in vain
Protein secretion, urea synthesizing, P450 enzymatic activitys）Evaluation, so as to evaluate the hepatotoxicity and pharmaceutical activity of medicine.In addition, medicine
Thing is dissolved in cell culture fluid by various concentrations, and it is empty to flow slowly into it via the import of liver organ many cells co-culture system
Intracavitary portion, medicine directly acts on hepatic parenchymal cells aggregation and many cells species, and metabolism via liver parenchymal cell and
Conversion, the evaluation of liver cell morphology characterization and cellular functional activity is carried out with this, so that it is thin to normal liver also to carry out medicine
Detection of the security or medicine of born of the same parents to the pharmaceutical activity of pathological liver cell.
The foregoing examples are merely illustrative of the technical concept and features of the invention, its object is to allow the person skilled in the art to be
Present disclosure can be understood and implemented according to this, it is not intended to limit the scope of the present invention.It is all smart according to the present invention
Equivalent transformation or modification that refreshing essence is done, should all be included within the scope of the present invention.
Claims (7)
1. a kind of microvascular liver chip based on cell aggregation, it is characterised in that：Including the microvasculature positioned at upper strata
（1）, middle class blood vessel endothelium barrier system（2）With the liver organ many cells co-culture system of lower floor（3）, microvasculature
（1）With liver organ many cells co-culture system（3）It is respectively arranged in respective chip base；Wherein, the microvasculature（1）
Including by some choked flow fence（4）The curved vessel that interlaced arrangement is constituted（5）, the curved vessel（5）Two ends are respectively equipped with micro-
Vascular import（6）With microvascular outlet（7）；The class blood vessel endothelium barrier system（2）By perforated membrane（8）Composition；The liver
Organ many cells co-culture system（3）Including cell aggregation enrichment region（9）Area is co-cultured with various kinds of cell（10）, and at two ends
It is respectively arranged with culture systems import（12）With culture systems outlet（11）, liver organ many cells co-culture system（3）It is located at
Culture systems import（12）Interior zone be provided with two shunting fence（13）, wherein, cell aggregation enrichment region（9）By many
Individual crescent moon groove-like structure（15）Composition, each crescent shape structure（15）On be provided with some gaps（14）, multiple crescents
Shape structure（15）Longitudinal arrangement is into several columns, and the several columns interlaced arrangement, all crescent moon groove-like structures（15）The equal court of opening
Culture systems import（12）；
The liver organ many cells co-culture system（3）Culture systems outlet（11）, culture systems import（12）Passage is deep
Degree, shunts fence（13）With crescent moon groove-like structure（15）Height be 100 μm；Shunt fence（13）For round and smooth arc, with training
Support system inlets（12）Passage alignment, and be equidistantly arranged symmetrically；Crescent moon groove-like structure（15）Number be 80, its outer toroid
Radius is 350 μm, and interior annular radius is 150 μm, crescent moon groove-like structure（15）On gap（14）For four, and uniformly diverging row
Row, its width is 20 μm, 100 μm of depth, and interior annular opening air line distance is 100 μm, crescent moon groove-like structure（15）Each other
Spacing distance be 100 μm.
2. the microvascular liver chip according to claim 1 based on cell aggregation, it is characterised in that：The chip base is
Dimethyl silicone polymer, polymethyl methacrylate, glass, makrolon, polytetrafluoroethylene (PTFE), nitrocellulose, biomembrane, silicon
The one or more of piece.
3. the microvascular liver chip according to claim 1 based on cell aggregation, it is characterised in that：The liver device
Official's many cells co-culture system（3）Cell adherence used and culture matrix are Fibrin Glue, extracellular matrix protein, BSA
Albumen, fibroin albumen, gelatin, chitosan, arginine-glycine-aspartic acid, matrigel, sodium alginate, different methacryl
The one or more of amine, polyethylene glycol or polyethylene glycol acrylate.
4. the microvascular liver chip according to claim 1 based on cell aggregation, it is characterised in that：The microvascular
System（1）Scarcely perceptible pulse tube inlet（6）With microvascular outlet（7）Channel depth, choked flow fence（4）Height and curved vessel（5）It is deep
Degree is 100 μm；Choked flow fence（4）Width be 20 μm, be shaped as semicircle chamfering, and co-culture with liver organ many cells
System（3）Middle crescent moon groove-like structure（15）Position it is corresponding；Curved vessel（5）It is round and smooth passage, channel width is 600 μ
M, number of bends is 20.
5. the microvascular liver chip according to claim 1 based on cell aggregation, it is characterised in that：The perforated membrane
(8) material is dimethyl silicone polymer, one kind of Transwell PET or PC films, and thickness is 10 μm, and aperture is
0.4,1,3,5,8 μm.
6. a kind of preparation method of any described microvascular liver chips based on cell aggregation of claim 1-5, it is special
Levy and be：Comprise the following steps：
（1）The mask plate of organ chip, micro- knot of each layer chip of accurate Drawing are designed and drawn with computer aided design software
Structure and microchannel figure；
（2）By the method for photoengraving, bilevel mask is prepared, the chip containing micro-structural and microchannel is prepared；
（3）By the microvasculature chip on upper strata, middle perforated membrane and the liver organ many cells co-culture system of lower floor enter
Row alignment, pressurization, and plasma bonding is carried out, form the micro-structural and microchannel network of 3 D stereo.
7. a kind of application method of any described microvascular liver chips based on cell aggregation of claim 1-5, it is special
Levy and be：
First, different cell categories are planted by microvascular import and culture systems intake channel successively, and carried out adherent and three
Aggregation culture is tieed up, and carries out liver parenchymal cell pattern and Evaluation of Functional；
During repopulating cell, plantation blood vessel endothelium is thin in the closed area of microvasculature and the formation of class blood vessel endothelium barrier system
Born of the same parents, cell density is 1x106Individual/mL, adhere-wall culture 2-4 hours forms vascular endothelial cell layer；It is common in liver organ many cells
Culture systems plant various kinds of cell successively, and liver cell aggregation enters cell aggregation by culture systems intake channel first
The crescent moon groove-like structure of enrichment region, enrichment forms close cell connection, and carries out three-dimensional aggregates growth；Various kinds of cell is trained altogether
Support area and co-cultured cell species is planted by culture systems intake channel, co-cultured cell species includes stellate cells, Ku Pu
Not cell, horn cell, and fibroblast, cell density is 1x106Individual/mL, adhere-wall culture 2-4 hours；
Then, it is passed through medicine and carries out medicine hepatotoxicity and pharmaceutical activity evaluation, includes the long-term and short-term hepatotoxicity of medicine,
And the IC50 values of medicine；
Finally, when carrying out drug evaluation, medicine passes through microvasculature（1）Into chip, class blood vessel endothelium screen is then penetrated
Barrier system（2）, enter the liver organ many cells co-culture system of lower floor（3）, so that evaluate the toxic action of its liver with
And carry out the metabolism and conversion of medicine；Or, medicine is also by culture systems import（12）It is directly entered liver organ many cells
Co-culture system（3）, directly contacted with hepatic parenchymal cells aggregation and co-cultured cell and realize metabolism and convert, and tested
Demonstrate,prove the hepatotoxicity and medicine vigor of medicine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510720578.9A CN105176816B (en) | 2015-10-30 | 2015-10-30 | A kind of microvascular liver chip and its preparation and application based on cell aggregation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510720578.9A CN105176816B (en) | 2015-10-30 | 2015-10-30 | A kind of microvascular liver chip and its preparation and application based on cell aggregation |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105176816A CN105176816A (en) | 2015-12-23 |
| CN105176816B true CN105176816B (en) | 2017-09-26 |
Family
ID=54899250
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510720578.9A Active CN105176816B (en) | 2015-10-30 | 2015-10-30 | A kind of microvascular liver chip and its preparation and application based on cell aggregation |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105176816B (en) |
Families Citing this family (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2602614B (en) * | 2017-03-10 | 2023-02-15 | Prellis Biologics Inc | Methods and systems for printing three-dimensional objects |
| US11085018B2 (en) | 2017-03-10 | 2021-08-10 | Prellis Biologics, Inc. | Three-dimensional printed organs, devices, and matrices |
| CN111032687A (en) | 2017-05-25 | 2020-04-17 | 普瑞利思生物制品公司 | Three-dimensional printed organs, devices and substrates |
| CN107502547A (en) * | 2017-09-25 | 2017-12-22 | 中科芯瑞(苏州)生物科技有限公司 | A kind of micro-fluidic chip for realizing various kinds of cell co-cultivation and its application |
| CN108004144B (en) * | 2017-12-18 | 2021-04-06 | 清华大学深圳研究生院 | Organ chip unit containing pipeline network |
| CN108504571B (en) * | 2018-03-09 | 2021-12-31 | 清华大学深圳研究生院 | Device and method for constructing artificial liver lobule functional unit |
| CN108641931A (en) * | 2018-04-04 | 2018-10-12 | 浙江大学 | A kind of digitlization microarray organ chip and its application |
| WO2019222871A1 (en) * | 2018-05-21 | 2019-11-28 | 深圳华大生命科学研究院 | Bionic intestinal-hepatic organ chip, preparation method therefor and application thereof |
| CN109385394A (en) * | 2018-11-02 | 2019-02-26 | 大连理工大学 | A kind of method of extracting and developing and the androgynous xenogenesis liver primary cell of culture |
| CN110387327A (en) * | 2019-07-10 | 2019-10-29 | 合肥学院 | A kind of three vascular lobuli hepatis chips |
| CN112264115B (en) * | 2020-10-26 | 2022-03-11 | 南京鼓楼医院 | Fishbone microfluidic chip carrying molecular imprinting inverse opal structure microspheres and preparation method thereof |
| CN112626025A (en) * | 2021-01-20 | 2021-04-09 | 温州医科大学附属第一医院 | Three-dimensional tumor cell drug resistance model and preparation method thereof |
| CN113362690B (en) * | 2021-05-31 | 2023-08-08 | 中国科学技术大学 | Liver small She Xinpian |
| CN114317272B (en) * | 2021-12-07 | 2024-01-12 | 上海前瞻创新研究院有限公司 | Culture device for multicellular co-culture and cell culture method |
| CN114276903A (en) * | 2021-12-24 | 2022-04-05 | 武汉大学 | Liver organoid culture chip, liver organoid model, and preparation method and application thereof |
| CN116445285A (en) * | 2023-03-28 | 2023-07-18 | 创芯国际生物科技(广州)有限公司 | Organ-like co-culture chip, construction method and co-culture method |
Family Cites Families (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1490520A4 (en) * | 2002-03-12 | 2006-06-07 | Surface Logix Inc | Assay device that analyzes the absorption, metabolism, permeability and/or toxicity of a candidate compound |
| CN101285036A (en) * | 2008-05-16 | 2008-10-15 | 深圳先进技术研究院 | Automatic cell culture microflow control chip device and method thereof |
| WO2012118799A2 (en) * | 2011-02-28 | 2012-09-07 | President And Fellows Of Harvard College | Cell culture system |
| US9513280B2 (en) * | 2012-08-28 | 2016-12-06 | University Of Utah Research Foundation | Microfluidic biological barrier model and associated method |
| CN104342369B (en) * | 2013-07-25 | 2017-08-25 | 国家纳米科学中心 | A kind of use micro-fluidic chip builds the device and its preparation and application of three-dimensional nerve network |
| CN103952300B (en) * | 2014-03-31 | 2017-07-28 | 大连医科大学 | A kind of micro-fluidic chip and cell chemotaxis motion study method |
| CN103981096B (en) * | 2014-05-27 | 2015-11-18 | 东南大学 | A kind of two-layer cell culture system organ chip and preparation method thereof |
| CN104164360B (en) * | 2014-07-29 | 2016-07-06 | 西北农林科技大学 | Integrated microfluidic chip and for three-dimensional nodule location, build, recovery method |
| CN104388299B (en) * | 2014-10-30 | 2016-03-16 | 东南大学 | A kind of micro-fluidic chip for cell capture |
-
2015
- 2015-10-30 CN CN201510720578.9A patent/CN105176816B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN105176816A (en) | 2015-12-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105176816B (en) | A kind of microvascular liver chip and its preparation and application based on cell aggregation | |
| Ashammakhi et al. | Gut-on-a-chip: Current progress and future opportunities | |
| CN112280678B (en) | Detachable and reusable hydrophobic or super-hydrophobic microfluidic organ chip | |
| CN103981096B (en) | A kind of two-layer cell culture system organ chip and preparation method thereof | |
| Amirifar et al. | Brain-on-a-chip: Recent advances in design and techniques for microfluidic models of the brain in health and disease | |
| CN102124096B (en) | Organ mimic device with microchannels and methods of use and manufacturing thereof | |
| CN110106081B (en) | Micro-fluidic chip for constructing brain function unit model and construction method | |
| CN105925480A (en) | Micro-fluidic chip for high-throughput screening of blood brain barrier drug permeability and preparation method of micro-fluidic chip | |
| CN106581761A (en) | Artificial liver tissue and preparation method thereof | |
| CN108277198A (en) | A kind of liver micro-fluidic chip and its application for realizing that two dimension, three dimensional intersection co-culture | |
| Park et al. | Reconstruction of in vivo-like in vitro model: enabling technologies of microfluidic systems for dynamic biochemical/mechanical stimuli | |
| Nguyen et al. | Microfluidic models of the human circulatory system: versatile platforms for exploring mechanobiology and disease modeling | |
| CN116445285A (en) | Organ-like co-culture chip, construction method and co-culture method | |
| Ahmed | Organ-on-a-chip microengineering for bio-mimicking disease models and revolutionizing drug discovery | |
| CN109456890A (en) | It is a kind of to be layered the band-like micro-fluidic chip for co-culturing 4 kinds of liver cells and its application | |
| CN109825437A (en) | A kind of micro-fluidic chip and cultural method for cell culture | |
| CN103981085A (en) | Self-set concentration gradient drug screening organ chip and preparation method thereof | |
| Yu et al. | Emerging strategies of engineering retinal organoids and organoid-on-a-chip in modeling intraocular drug delivery: Current progress and future perspectives | |
| CN106929417A (en) | A kind of multi-layer cellular culture micro element bionical based on vein eyed structure | |
| Hou et al. | Application of microfluidic chips in the simulation of the urinary system microenvironment | |
| CN110511866A (en) | A kind of multiple organ chip and its preparation method and application | |
| CN109554278A (en) | A kind of organ chip and the nano particle lung surface active oxidant layer interaction evaluation method based on organ chip technology | |
| CN220166205U (en) | Organ-like co-culture chip | |
| CN110408538A (en) | A kind of liver chip of more lobuli hepatis integrated morphologies | |
| CN212077076U (en) | Micro-fluidic experimental board |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |